D Liebowitz

Latent Epstein-Barr virus (EBV) infection and growth transformation of B lymphocytes is characterized by EBV nuclear and membrane protein expression (EBV nuclear antigen [EBNA] and latent membrane protein [LMP], respectively). LMP1 is known to be an oncogene in rodent fibroblasts and to induce B-lymphocyte activation and cellular adhesion molecules in the EBV-negative Burkitt's lymphoma cell line Louckes. EBNA-2 is required for EBV-induced growth transformation; it lowers rodent fibroblast serum dependence and specifically induces the B-lymphocyte activation antigen CD23 in Louckes cells. These initial observations are now extended through an expanded study of EBNA- and LMP1-induced phenotypic effects in a different EBV-negative B-lymphoma cell line, BJAB. LMP1 effects were also evaluated in the EBV-negative B-lymphoma cell line BL41 and the EBV-positive Burkitt's lymphoma cell line, Daudi (Daudi is deleted for EBNA-2 and does not express LMP). Previously described EBNA-2- and LMP1-transfected Louckes cells were studied in parallel. EBNA-2, from EBV-1 strains but not EBV-2, induced CD23 and CD21 expression in transfected BJAB cells. In contrast, EBNA-3C induced CD21 but not CD23, while no changes were evident in vector control-, EBNA-1-, or EBNA-LP-transfected clones. EBNAs did not affect CD10, CD30, CD39, CD40, CD44, or cellular adhesion molecules. LMP1 expression in all cell lines induced growth in large clumps and expression of the cellular adhesion molecules ICAM-1, LFA-1, and LFA-3 in those cell lines which constitutively express low levels. LMP1 expression induced marked homotypic adhesion in the BJAB cell line, despite the fact that there was no significant increase in the high constitutive BJAB LFA-1 and ICAM-1 levels, suggesting that LMP1 also induces an associated functional change in these molecules. LMP1 induction of these cellular adhesion molecules was also associated with increased heterotypic adhesion to T lymphocytes. The Burkitt's lymphoma marker, CALLA (CD10), was uniformly down regulated by LMP1 in all cell lines. In contrast, LMP1 induced unique profiles of B-lymphocyte activation antigens in the various cell lines. LMP1 induced CD23 and CD39 in BJAB; CD23 in Louckes; CD39 and CD40 in BL41; and CD21, CD40, and CD44 in Daudi. In BJAB, CD23 surface and mRNA expression were markedly increased by EBNA-2 and LMP1 coexpression, compared with EBNA-2 or LMP1 alone. This cooperative effect was CD23 specific, since no such effect was observed on another marker, CD21.(ABSTRACT TRUNCATED AT 400 WORDS)

A latent infection membrane protein (LMP) encoded by the Epstein-Barr virus (EBV) genome in latently infected, growth-transformed lymphocytes alters the phenotype of a human EBV-negative B-lymphoma cell line (Louckes) when introduced by gene transfer. These LMP-expressing cells exhibit increased homotypic adhesion due to increased expression of the adhesion molecules LFA-1 and ICAM-1. Increased homotypic adhesion could foster B-cell growth by facilitating autocrine growth factor effects. LFA-3 expression is also induced. The induction of LFA-3 and ICAM-1 results in increased heterotypic adhesion to T lymphocytes. This could result in more effective T-cell immune surveillance. Since LMP is expressed in EBV-transformed lymphocytes and has been demonstrated to transform rodent fibroblasts in vitro, a wide range of possible effects on B-lymphoma cell growth were assayed. In the Louckes B-lymphoma cell line, EBV LMP causes increased cell size, acid production, plasma membrane ruffling, and villous projections. Although cell proliferation rate was not greatly affected, the steady-state intracellular free calcium level, transforming growth factor beta responsiveness, and expression of the lymphocyte activation markers (CD23 and transferrin receptor) were increased. Thus, LMP appears to be a mediator of EBV effects on B-cell transformation. In transfected lymphoma cells, LMP localizes to patches at the cell periphery and associates with the cytoskeleton as it does in EBV-transformed B lymphocytes or in rodent fibroblasts. A partially deleted form of LMP (D1LMP) does not aggregate in patches or associate with the cytoskeleton and had little effect on B-cell growth. Thus, cytoskeletal association may be integral to LMP activity.

Epstein-Barr virus is known to encode three nuclear proteins and one membrane protein (LMP) in latently infected growth-transformed cells. Studies of the plasma membrane localization and orientation of LMP by protease digestion of live cells and by immunofluorescence indicated the following. (i) At least 30% of LMP is in the plasma membrane, as opposed to other cytoplasmic membranes. (ii) A small LMP domain which corresponds to a previously proposed outer reverse turn between the first two transmembrane domains is exposed on the outer cell surface (and two other proposed outer-reverse-turn domains may be exposed), whereas all or almost all of the rest of the protein is not exposed on the outer cell surface. (iii) LMP is present in patches in the cell plasma membrane.

The etiology of NPC is multifactorial and includes virological, genetic, and environmental factors as described. These factors can be synthesized into a model for the development of NPC through a multistep process. First, an individual may carry a genetic predisposed risk of developing NPC as suggested by the HLA linkage of the disease. The nasopharyngeal epithelium becomes infected early in life by EBV and viral gene expression eventually becomes limited to EBNA-1, LMP1, LMP2A/2B, and perhaps another viral protein not yet fully characterized. LMP1 has profound growth stimulating effects in vitro and may exert similar effects in the nasopharyngeal epithelium. As cells are stimulated to divide, the presence of EBNA-1 ensures that the viral genome will replicate and be distributed to progeny cells. Finally, to reach their full malignant potential, the dividing nasopharyngeal cells may acquire cellular genetic changes involving recessive TSGs on chromosome 3. The risk for developing secondary genetic alterations involving these genes may be increased by exposure to environmental carcinogens such as volatile nitrosamines in salted fish. Thus, NPC provides a model system for understanding the interactions of genetic, infectious, and environmental factors involved in oncogenic transformation. Recent developments in organoculture systems and transgenic animal technology should allow dissection at the molecular level of the specific mechanisms involved in this process.

The structures of the 2.3- and 2.0-kilobase Epstein-Barr virus (EBV) mRNAs, partially encoded within the EcoRI J fragment DNA of the viral genome, were determined by analysis of their cDNAs. Both mRNAs are transcribed across the fused terminal repeats of the EBV episome and consist of nine exons. The mRNAs are transcribed from different promoters and have a unique 5' exon from the U5 region of the genome but eight common exons from the U1 region. One principal open reading frame is present in each mRNA and is predicted to encode 54,000- and 40,000-dalton integral membrane proteins. This result was confirmed by in vitro translation of RNAs in the presence of canine pancreatic microsomes. The 2.3-kilobase mRNA is not expressed in Raji cells, owing to the deletion of the 5' regulatory and coding region of this gene, whereas neither mRNA is expressed in Namalwa cells, owing to inactivation as a result of integration of the EBV genome via the terminal repeats. Since these mRNAs are readily detected in largely latently infected cells and do not increase in abundance with EBV replication, these putative latent-infection membrane proteins are tentatively designated LMP-2A and LMP-2B, respectively.